Microscopes have been used for the observation of organic and non-organic materials for a long time and in a multitude of applications. The resolution attainable with light microscopes was diffraction limited throughout a significant portion of that period.
Only recently, techniques were developed that allow high-resolution microscopy to be performed, providing a resolution that is less, or even much less, than the diffraction limit. Examples for such techniques include techniques that employ a structured illumination of the object, or luminescence microscopy methods in which objects may be localized with a high precision. The article by L. Schermelleh et al., “A guide to super-resolution fluorescence microscopy”, J. Cell Biol. 190(2): 165 (2010), provides an overview over such techniques. For further illustration, such techniques are also described in patent publications European Patent EP 1 157 297 B1 or German Published Patent Application DE 10 2006 021 317 B3. Using such techniques, it has become possible to localize objects with a precision in the range from a few nanometers to a few tens of nanometers. Methods that allow high-resolution luminescence microscopy to be used for three-dimensional imaging are described in German Published Patent Applications DE 10 2009 043 744.4 entitled “Method and microscope for performing three-dimensional resolution-enhanced microscopy”, DE 10 2009 060 490.1 entitled “High-resolution microscope and image splitter arrangement”, and DE 10 2009 060 793.5 entitled “High-resolution microscope and method of determining object positions in two or three dimensions”, all of which are assigned to the assignee of the subject application.
A characteristic of various microscopy methods that allow a resolution much smaller than the diffraction limit to be attained is that data acquisition and data processing may be complex. In data acquisition, a plurality of individual two-dimensional (2D) frames of the object may be captured, which may be processed to compute resulting image data of the object therefrom. As an illustration, in methods that employ a structured illumination of the object, different orientations of the illumination pattern may be used, and one or plural 2D-frames may be captured for each one of the different orientations. The image of the object is generated by processing the information contained in the various 2D-frames using a computer.
For further illustration, high-resolution luminescence microscopy method may use molecules that are optically switchable or optically activatable. To ensure that, for each captured frame, the molecules that can be detected are not located at too small a distance from each other, only a small fraction of the molecules is switched to a state in which it can be detected for each one of the individual captured 2D-frames. A large number of frames, such as from 10,000 to 20,000 frames, may be required to combine the frames to a representation of the examined object that is essentially complete.
Such high-resolution methods may allow values for adjustable parameters to be set in a user-defined manner in order to further enhance the resolution of the resulting image that is obtained by computationally processing the raw data. For methods that employ a structured illumination, examples for such adjustable parameters include the illumination pattern (e.g., the periodicity of the pattern) or the number of different orientations of the illumination pattern for which frames are captured. For methods using luminescence microscopy, examples for such adjustable parameters include a fitting mask that is used to computationally evaluate a frame for localizing luminescence events or a spectrum and intensity of switching signals. For methods in which frames are captured in different object planes, the adjustable parameter may include the positions of the object planes, measured along the axial direction of the microscope, for example. Further examples of adjustable parameters include the characteristics of filters that are used to filter images in position space or in Fourier space.
Due to the complexity of the procedure upon which the data acquisition and data processing is based, it may be difficult for a user to understand the importance and affect of the various adjustable parameters for the underlying procedure. Understanding such affects would be important for the user in particular at the time at which the user sets the values for the adjustable parameters to carry out an experiment or to process raw data.
The user's understanding of the importance of the various adjustable parameters may be aided, to a certain degree, by methods that illustrate the affect of an adjustable parameter on the resulting image. Such simulation methods may use data that have previously been captured or exemplary data stored in a memory. German Published Patent Application DE 10 2007 046 469 A1 describes an example for such a method. Performing data processing using the values that are presently set for the various adjustable parameters may help the user identify suitable settings for the structure that is being examined at the time. However, no information on the underlying procedure is conveyed thereby. Such information would be particularly valuable to assist a user in understanding the importance and affect of the various adjustable parameters, and to use this understanding when data acquisition and data processing is performed for a new object. Moreover, when a simulation of data processing of captured data is performed using a method as described in German Published Patent Application DE 10 2007 046 469 A1, raw data must first be captured. The results of the simulation only have limited value for planning data acquisition. In particular, in cases in which data acquisition may be performed on a given sample only one time, as may be the case when molecules are optically switched in an irreversible manner in a luminescence microscopy data acquisition, it would be desirable for the user to also have a better understanding of the importance and affect of the various adjustable parameters on data acquisition.
There is a need for a microscope system, a microscopy method, and a storage medium storing computer-executable instruction code that address some of the above shortcomings. There is a need for systems and methods that assist a user in planning data acquisition and/or data processing. There is a need for systems or methods that provide the user with information on the affects that various adjustable parameters have when microscopy is performed, even when the procedure for data acquisition and/or data processing is complex.
Thus, a need exists to overcome the problems with the prior art systems, designs, and processes as discussed above.